Prostaglandin F2α Causes Fast Degenerative Changes in Ovulated Mouse Oocytes.

The effects of prostaglandin F2α on the cytoskeleton and membrane organelles of oocytes was investigated by culturing ovulated mouse oocytes in its presence (50 or 100 ng/ml) for 3 h. Tubulin, fibrillar actin, membranes and chromatin were visualized by specific antibodies, phalloidin, lipophilic dye DiOC6 and Hoechst 33342, respectively. Control oocytes were characterized by a meiotic spindle with chromosomes aligned at its equator, and a cortical layer of microfilaments with an actin cap. Intracellular membranes were localized mostly in the central region in metaphase I and in a broader volume, but still excluding the cell periphery, in metaphase II, and were slightly concentrated around the chromosomes. In oocytes treated with 50 ng/ml prostaglandin, cortical actin staining was diminished, the membrane distribution was clustered, and chromosomes showed signs of misalignment despite the apparently preserved spindle. In cells treated with 100 ng/ml prostaglandin, both the spindle and the actin cortex had degenerated or disappeared as microscopic objects. Metaphase plates were on average broader and more disorganized than in the 50 ng/ml group, and the distribution of membrane organelles had become uniform. These effects, to our knowledge observed for the first time, did not require presence of the cumulus during the incubation. They could be regarded as acceleration of the oocyte postovulatory aging, in which cytoskeletal deterioration seemed to have a leading role.

A retrospective analysis of adverse effects of an in vivo fluoroquinolone antibiotic enrofloxacin treatment on oocyte quality in the common marmoset.

Here we report a retrospective analysis of negative effects of routine enrofloxacin treatment of recurrent diarrhea on the ovary and the developing oocytes of the common marmoset, a small New World primate. The most deleterious effect on oocytes was observed about two months post treatment suggesting that the enrofloxacin effect is on early growing follicles. Manifestations of toxicity included decreased numbers of growing follicles and recovered culturable oocytes, as well as signs of early atresia of granulosa cells. In addition, increased amounts of holed stroma after treatment strongly suggested increased death of the early growing follicles. Of the oocytes judged to be of adequate quality for culture, maturation rates were not affected but fertilization of in vitro matured MII oocytes and subsequent cleavage rates were severely reduced in the enrofloxacin treated animals. Further, the arrested oocytes, which failed to mature or fertilize, showed obvious meiotic spindle abnormalities.

Dynamics of Lamins B and A/C and Nucleoporin Nup160 during Meiotic Maturation in Mouse Oocytes.

This study was aimed at elucidating the fate of three important nuclear envelope components - lamins B and A/C and nucleoporin Nup160, during meiotic maturation of mouse oocytes. These proteins were localized by epifluorescence and confocal microscopy using specific antibodies in oocytes at different stages from prophase I (germinal vesicle) to metaphase II. In immature germinal vesicle oocytes, all three proteins were detected at the nuclear periphery. In metaphase I and metaphase II, lamin B co-localized with the meiotic spindle, lamin A/C was found in a diffuse halo surrounding the spindle and to a lesser degree throughout the cytoplasm, and Nup160 was concentrated to the spindle poles. To our knowledge, this is the first report on nucleoporin localization in mammalian oocytes and the first successful detection of lamins in mature oocytes. While the distribution patterns of both lamins closely paralleled the respective stages of mitosis, Nup160 localization in metaphase oocytes corresponded to that in mitotic prometaphase rather than metaphase. The peculiar distribution of this nucleoporin in oocytes may reflect its role in meiosis-specific mechanisms of spindle assembly and its regulation.

Critical estradiol dose optimization for oocyte in vitro maturation in the common marmoset.

The aim of the present study was to critically evaluate the effect of different concentrations of estradiol (E2) during IVM of common marmoset (Callithrix jacchus) oocytes from antral follicles. The doses tested were 0, 0.1, 1, or 10 µg/mL E2 (referred to as 0 E2, 0.1 E2, 1 E2, and 10 E2 groups). After a preincubation, the concentration of E2 in IVM drops under oil was approximately 20% of the amount added (0.02; 0.2 and 1.9 µg/mL, respectively) because of absorption into the oil. Oocyte progression to metaphase II was significantly higher in the 0.1 E2 group than that in the absence of E2. With progressively higher doses, the maturation rate tended to decrease suggesting an overdose effect. Furthermore, the total first cleavage rate was significantly higher in the 0.1 E2 group than that in the 0 E2 group and decreased progressively with further increases in E2 concentration, with the 10 E2 group showing the same low rate as without E2. The oocytes which failed to cleave, after maturation in 10 E2, showed obvious signs of overdose with the highest rates of degeneration and abnormal spindle form, and an absence of embryo progression. In contrast to these obvious negative effects on the oocyte, 10 E2 was the only group in which a significant increase in radial cumulus expansion was observed. The concentration 0.1 E2, which is 10 times lower than the most commonly used E2 dose, produced the best results in all oocyte factors evaluated. These results represent the first study for a primate species showing a strong positive effect of E2 on oocyte maturation and embryo development, but only at the optimal concentration, and emphasize the critical limits of the optimal concentration range.

Numerical chromosome disorders in the common marmoset (Callithrix jacchus)--comparison between two captive colonies.

BACKGROUND: Chromosomal analyses were performed for marmosets from two colonies - Deutsches Primatenzentrum (DPZ) and Biomedical Primate Research Centre (BPRC). Chlorine-based disinfectants are used in DPZ; no chemical disinfection is applied in BPRC. METHODS: The rates of chromosomal non-disjunction, polyploidy and endoreduplication were investigated after G-banding. RESULTS: For DPZ monkeys, the mean rates of non-disjunction were 7.6% for bone marrow and 11.3% for lymphocytes. The polyploidy level was 2.5% in bone marrow and 0.8% in blood. Frequency of endoreduplication in bone marrow and in leucocytes was 0.5% and 0.8%, respectively. For BPRC, the rate of non-disjunction in leucocytes (1.3%) was significantly lower than that for DPZ; the polyploidy rate (0.2%) in blood was lower than that in DPZ; endoreduplication was not observed. CONCLUSION: The levels of chromosomal disorders are elevated for DPZ colony. We suggest that the increased rate of chromosomal disorders in DPZ marmosets can be related to the chemical disinfection of their environment.

[Investigation of cytokeratin and vimentin intermediate filaments in polycystic ovaries (PCOS) -- presence and specific structure of Balbiani body in primordial follicles].

Despite of the importance of cytoskeletal proteins for ovarian function and pathology, very few studies have addressed the presence and distribution of these proteins in polycystic ovaries. We investigated sections of human polycystic ovarian tissue for vimentin and a set of cytokeratins by epifluorescence. The studied proteins showed strong colocalization. Positive reaction was detected in two main ovarian compartments: with weak intensity in follicular cells and very strong in perinuclear position in oocytes of primordial follicles. Epifluorescent study of the oocytes from primordial follicles allowed us to identify the immunopositive structure in oocytes as Balbiani body, a transient organelle responsible for establishing oocyte polarity and ooplasm gradients in nonmammalian vertebrates. Our results suggest functional interaction of different types of cytoplasmic intermediate filament proteins in polycystic ovaries and a possible importance of the Balbiani body for human oogenesis in norm and pathology.

Variations of chromatin, tubulin and actin structures in primate oocytes arrested during in vitro maturation and fertilization--what is this telling us about the relationships between cytoskeletal and chromatin meiotic defects?

A nonhuman primate model was applied to investigate the relationships between variations in the organization of microtubules, microfilaments, and chromatin in metaphase I and metaphase II oocytes. Marmoset oocytes were subjected to in vitro maturation and coincubation with sperm. Oocytes which failed to cleave were investigated for chromatin, tubulin, and actin using Hoechst 33258, fluorescein isothiocyanate (FITC)-labeled alpha-tubulin antibody and rhodamine-labeled phalloidin, respectively. Spindles were categorized according to size, shape and microtubule organization: normal, large, multipolar, disorganized, absent spindle, and spindles with broad poles. Actin caps were categorized as: normal, small, split, and disorganized. Chromosomal condensation and alignment were described as normal or abnormal. Improper chromosomal condensation was associated with both abnormal microfilament and microtubule arrangement. This was further associated with abnormal actin organization, disorientation and late stabilization of microtubules, but not related to abnormal organization of spindle poles. Chromosomal misalignment was associated with disorientation and late stabilization of tubulin, but not to broad spindle pole. Additionally, abnormal actin polarization appeared not to be related to abnormal spindle poles. The model system presented in this study could be used as an experimental platform for studying the contribution of different factors to the exactness of late meiotic events in primate oocytes. The present study provides basic information on spindle, chromosome, and actin normal and abnormal organization, which can be observed in in vitro matured, but failed to cleave primate oocytes.

Epidermal growth factor effects on marmoset monkey (Callithrix jacchus) oocyte in vitro maturation, IVF and embryo development are altered by gonadotrophin concentration during oocyte maturation.

BACKGROUND: This is the first study of the effect of epidermal growth factor (EGF) on marmoset monkey oocytes matured in vitro. METHODS: We have evaluated the effects of 10 ng/ml EGF in combination with 1 or 10 IU/ml of gonadotrophins (FSH/hCG 1:1 ratio) during in vitro maturation (IVM) of marmoset oocytes. Immature cumulus-oocyte complexes (COCs) were retrieved from ovarian antral follicles of unprimed monkeys. COCs from six animals (n= 268) used in this study were randomly distributed among four experimental groups: (A) 1 FSH +1 hCG; (B) 10 FSH +10 hCG; (C) 1 FSH +1 hCG + EGF; and (D) 10 FSH +10 hCG + EGF (where 1 and 10 are concentrations, IU/ml). After IVM, oocytes were fertilized in vitro and embryos were allowed to progress up to 87-88 h. RESULTS: the highest rate of total and radial cumulus expansion was observed in Group A, with the lowest in Group B (P < 0.05). Neither maturation nor fertilization rate were affected by gonadotrophin concentration or presence of EGF. Addition of EGF increased degeneration and decreased first cleavage rate, which was significantly lower in Group C than Group A (P < 0.005). Interestingly, in the EGF groups some embryos cleaved faster than without EGF. CONCLUSIONS: The effects of EGF are highly dependent on concentration of gonadotrophins present in IVM medium. EGF has a negative effect on oocytes in the presence of low gonadotrophins, but contrastingly partially protects oocytes from the negative effects of high gonadotrophins. We propose that these observed negative effects of EGF may suggest use of an inappropriate dose of growth factor.

[Role of oocyte cytoplasmic factors in human IVF failure].

Premature chromosome condensation (PCC) of the sperm chromatin and abnormal dispersion of oocyte chromosomes are independent indirect indicators for cytoplasmic immaturity and cytoskeletal anomalies in the oocyte affecting the normal zygote and embryo formation in human IVF-ET practice. In a group of 66 human unfertilized oocytes, two types of cytoplasmic factors were registered: PCC in 49 (74.24%) and cytoskeletal anomalies in 17 oocytes (25.76%). These data were analyzed according to the main stimulation regimen and female age as well as to the familial factor of infertility. Our results displayed a higher proportion of PCC if the stimulation regimen included gonadotrophins alone (77.55%) compared with the combined GnRH agonist and gonadotrophins administration (22.45%) and in the female age group of 36-40 years (63.33%). Cytoskeletal defects were predominant in the female age group of 31-35 years (46.06%). Remarkable was the significance of cytoplasmic factors in cases of unknown infertility factor--oocytes from these patients included 34.69% of all cells with PCC and 52.94% of all cells with cytoskeletal defects. Hence, these anomalies were probable causes for IVF failure in cases of unexplained infertility Conclusions: 1. The main regimen for hormonal stimulation and the female age influence the cytological status of studied oocytes (cytoplasmic immaturity causing PCC and polyspermy). 2. Cytoplasmic factors (immaturity and cytoskeletal anomalies) are a prognostic factor for the IVF success in cases of unknown infertility problem in the family.

Meiotic abnormalities in in vitro-matured marmoset monkey (Callithrix jacchus) oocytes: development of a non-human primate model to investigate causal factors.

BACKGROUND: Meiotic abnormalities are thought to be a major causal factor of low embryo development rates, for embryos developed from in vitro-matured oocytes. A new non-human primate model, in the common marmoset, is being developed to facilitate investigation of the mechanisms involved. METHODS: Oocytes were dissected from antral follicles from three size classes. They were allowed to mature in vitro for only 24 h, in order to focus the investigation on the rapidly maturing oocytes. Chromosome spreads were visualized with Giemsa staining, and spindles /chromosomes with fluorescently labelled anti-alpha-tubulin antibody combined with a DNA fluorochrome. RESULTS: 40% of the oocytes had reached metaphase II (MII) after 24 h. Of the MII oocytes selected for karyotyping, readable chromosomal spreads were obtained from 64%. Overall, 63% of these presented a normal haploid chromosome number of 23,X, with all abnormal karyotypes occurring in the oocytes from small follicles. For another group of MII oocytes, where meiotic spindles were visualized, only half of the MII oocytes displayed well-formed spindles and apparently correct chromosomal alignment. CONCLUSIONS: This work provides the first information on the normal and aneuploid MII meiotic chromosome sets for the marmoset oocyte, and demonstrates a high rate of chromosomal and spindle abnormality among rapidly maturing oocytes from small antral follicles.

The status of the chromatin of human preimplantation embryos with good morphology.

The data about the relation and succession of blastomere fragmentation, cleavage rate and chromatin anomalies in preimplantation mammalian embryos are empirical and controversial at present. In this work we studied the proportion of nuclear fragmentation and condensation in 3-5-cell stage human embryos with no or minimal blastomere fragmentation (morphological class A and B, respectively) and the possibilities to perform FISH chromosomal analyses with them. We observed different stages of chromatin damage in blastomere nuclei corresponding to the steps of nuclear apoptotic changes well known in many cell types. The ploidity analysis of chromosomes 1, 5, 19 and X was determined as successful in embryos which had at least 2 out of 3, 3 out of 4 or 3 out of 5 normal nuclei with an equal number of FISH signals. There was no difference in the percentage of abnormal nuclei among the A- and B-class embryos. Tendencies noted by us suggest that the minimal blastomere fragmentation (up to 20% of perivitelline space) does not preclude the normal nuclear status allowing successful ploidy testing. The presence of condensed chromatin is a critical factor for interphase cytogenetic analysis of single early blastomeres.

Supernumerary human preembryos provide potential for preimplantation genetic diagnosis.

Preimplantation genetic diagnosis is an alternative to the classical prenatal diagnosis for couples undergoing in vitro fertilization. It allows very early embryo selection--before the intrauterine embryo transfer. Prior to clinical application of preimplantation diagnosis in the Infertility Treatment Centre "Technobioassistance", Sofia Medical Faculty, we have developed preimplantation diagnosis models of human spermatozoa and untransferred 2-8-cell human embryos obtained in vitro. Directly fluorescein isothiocyanate-labelled probes specific for the centromeric regions of chromosomes 1, 5, 19 and X (Boehringer Mannheim) were used. Eighty-six point three percent of fixed blastomeres with normal size and shape had unfragmented nuclei with dispersed interphase chromatin or mitotic chromosomes and all of them demonstrated successful hybridization. In cases with more than 75% of embryo cells suitable for analysis we were able to estimate the presence of mosaicism among the blastomeres.